The generation of DNA sequence information has dramatically increased due to the large programs for sequencing of the human genome. Today that work is mainly done by traditional gel electrophoretic separation of DNA fragments terminated at different positions, either enzymatically (dideoxy chain termination method according to Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977)) or chemically (chemical degradation method according to Maxam and Gilbert, Proc. Natl. Acad. Sci. USA 74: 560-564). These systems are, however, both time- and labour-intensive.
There is therefore a general need for more effective methods for de novo sequencing of DNA as well as for repeated sequencing of known sequences for analysis of mutations, such as point mutations. The mutation analysis will increase as more information will be gathered about the correlation between different diseases and mutations and also due to the need to verify deliberately introduced mutations in biotechnology work.
Sequencing by hybridization (SBH) (see e.g. Drmanac et al., Genomics 4: 114; Strazoski et al., Proc. Natl. Acad. Sci. USA 88: 10089 (1991); Bains and Smith, J. Theoretical Biol. 135: 303 (1988); and U.S. Pat. No. 5,202,231) has become an interesting alternative to traditional sequencing with a potential for higher through-put of information. This type of system utilizes the information obtained from multiple hybridizations of the polynucleotide of interest, using short oligonucleotides to determine the nucleic acid sequence. However, there are several technical problems associated with this technology. For example, while today there are ways to build arrays of oligonucleotides on a chip based on the synthesis of oligoprobes and photolitographic techniques, it is still complicated to provide on a chip the large set of oligonucleotide probes required for determining a random nucleic acid sequence. Further, the detection of interaction of labelled target DNA is normally done by fluorescent or radioactivity measurements. Such detection is dependent on washing of the chip to get rid of residual labelled target molecules and the oligoprobes must therefore bind rather strongly to the target molecules. There are also problems with the binding of oligoprobes with a single base mismatch in combination with the different sensitivity to washing conditions dependent on base pair composition, G:C being more stable than A:T. One attempt to overcome such problems is to use tetraalkylammonium salts that eliminate the difference in stability of G:C and A:T base pairs.
Even if differences in base composition can be compensated for, the whole SBH procedure is based on interaction, washing, and detection of hybridized target DNA and oligoprobe. The conditions for the hybridization thus have to be adjusted for a stable hybridization which can be detected only after several washing cycles. Dependent on the position of the mismatch of single bases, base composition, oligoprobe length and temperature, there will be several hybridizations of oligomers that will show up as weaker binding and such interactions will be problematic to determine. Temperature and salt gradients elution have been suggested but are difficult to elaborate technically.
Due to the conditions needed for hybridization there is also always a potential risk for the target DNA to hybridize to itself due to complementary regions of the DNA.
A major disadvantage of SBH is, however, that the information is exclusively based on short-range information and the fact that overlaps are unique. Success is dependent on whether or not there are repeated sequences in the nucleic acid to be analysed. The need and importance of repeated sequences are known from several situations, not least in the analysis of genes like, for example, the gene for Huntington's disease where repeated sequences and the amount of repeats have clinical relevance.
For the analysis of known sequences or of a particular site, mutation analysis may be advantageous. An example of this is the mutation dependent tumour frequency found for proteins such as p53. Binding of p53 to DNA is crucial for a correct control of cell growth and mutation dependent methods for therapy are likely to be developed. Furthermore, the kind of mutation detected may affect the treatment and aid in selecting the appropriate drug.
Label-free real-time measuring techniques, such as those based on surface plasmon resonance (SPR), have been used to study the hybridization of DNA and oligomeric probes (oligoprobes). Attempts have also been made to analyse the kinetic information of the hybridization to determine the degree of hybridization, e.g. to detect mutant sequences, as described in Biosensor Application Note 306, 1994, Pharmacia Biosensor AB, Sweden. It has, however, been found that such analyses are difficult to use for obtaining relevant mismatch information as the kinetics for hybridization is complex under the conditions for hybridization normally used, which result in the formation of relatively stable hybridization complexes with long half-lives.
WO 93/25909 discloses the use of label-free techniques mentioned above in combination with immobilised receptors, specifically antibodies, which are selected or designed to have a high dissociation constant, or "off-rate", for the binding of analyte to the receptor. Such a detection system will rapidly respond to changes in the analyte concentration and regeneration of the sensing surface supporting the receptor will not be required. Typically, the receptor is selected such that the dissociation rate constant (k.sub.off or k.sub.diss) for a particular analyte of interest is higher than 10.sup.-2 per second.
WO 95/00665 discloses a method of providing the sequence of a single stranded nucleic acid molecule, which when hybridized to a complementary single stranded molecule results in a double (duplex) structure having a preselected value for a free energy parameter, such as ligand binding, melting temperature or affinity for a target sequence. Thereby nucleic acid molecules may be produced which are tailored for specific applications, e.g. nucleic acid molecules with a defined affinity for a ligand which binds to the DNA and regulates the expression of a protein encoded by the nucleic acid.